Syringin inhibits endogenous volume regulated anion channel currents of HEK293 cells in hypotonic circumstances

Abstract Syringin is a natural chemical compound first isolated from the bark of lilac and is known to have neuroprotective effects in middle cerebral artery occlusion (MCAO). Volume regulated anion channel (VRAC) is a cell swelling‐activated anion channel, which is implicated in brain ischemia. However, the mechanism underlying the syringin protecting the neuron from damage in MCAO is still unclear. We hypothesized that syringin has an inhibitory effect on the opening of VRAC channels. To access the effect of syringin on VRAC currents and predict how syringin interacts with VRAC proteins, we performed whole‐cell patch‐clamp experiments using HEK293 cells. Initially, HEK293 cells were perfused with isotonic extracellular solution, followed by hypotonic extracellular solution to stimulate endogenous VRAC currents. Once the VRAC currents reached a steady state, the hypotonic solution containing syringin was perfused to study the effect of syringin on VRAC currents. The potential interaction between syringin and the VRAC protein was investigated using molecular docking as a predictive model. In this study, we found that syringin moderately inhibited VRAC currents in a dose‐dependent manner. The potential binding of syringin to LRRC8 protein was predicted through in silico molecular docking, which suggests an affinity of −6.6 kcal/mol and potential binding sites of arginine 103 and leucine 101. Our results herein characterize syringin as an inhibitor of the VRAC channels, which provides valuable insights for the future development of VRAC channel inhibitors.

Initially, HEK293 cells were perfused with isotonic extracellular solution, followed by hypotonic extracellular solution to stimulate endogenous VRAC currents. Once the VRAC currents reached a steady state, the hypotonic solution containing syringin was perfused to study the effect of syringin on VRAC currents. The potential interaction between syringin and the VRAC protein was investigated using molecular docking as a predictive model. In this study, we found that syringin moderately inhibited VRAC currents in a dose-dependent manner. The potential binding of syringin to LRRC8 protein was predicted through in silico molecular docking, which suggests an affinity of −6.6 kcal/mol and potential binding sites of arginine 103 and leucine 101. Our results herein characterize syringin as an inhibitor of the VRAC channels, which provides valuable insights for the future development of VRAC channel inhibitors.

K E Y W O R D S
inhibitor, molecular docking, syringin, VRAC channels

| INTRODUC TI ON
Volume regulated anion channel (VRAC) is an anion channel ubiquitously expressed in the membranes of vertebrate cells. Changes in osmotic pressure can cause cells to swell, and the process of regulatory volume decrease (RVD) is responsible for restoring the abnormal cell volume. VRAC channels play a critical role in RVD by facilitating the regulated passive efflux of chloride and small organic compounds, thereby enabling water to exit the cytoplasm. 1 In addition to transferring the inorganic ion and organic osmolytes, VRAC channel also mediates glutathione, cGAMP, and anti-cancer drugs. 2-4 VRAC channel has been identified as a heteromeric complex composed of LRRC8 protein family (LRRC8A, LRRC8B, LRRC8C, LRRC8D, and LRRC8E). Of these proteins, LRRC8A is the sole essential subunit required for functional VRAC channel formation, but it must also associate with at least one of the other LRRC8 proteins to form a functional channel. 5,6 The hexameric assembly of LRRC8 proteins results in a diverse range of VRAC complexes with different subunit compositions. 5,7,8 High-resolution cryo-EM structures for homomeric LRRC8A were revealed in 2018 by three distinct research teams. [9][10][11] The LRRC8 protein contains 4 transmembrane domains (TM1-TM4), extracellular domain (EC), intracellular linker domain (IL), leucine-rich repeat domain (LRR), N-terminus and C-terminus located inside the cell. The VRAC currents display characteristic features, including moderate outward rectification and inactivation tendency under cytoplasmic positive potentials. [12][13][14] Cell swelling is relevant to ischemia-induced brain damage.
Astrocytes are susceptible to swelling in the central nervous system injury, and this process is mediated by the opening of VRAC channels.
The activation of VRAC channels facilitates excitatory glutamate efflux from astrocytes, resulting in neuronal damage. Studies have shown that defects in astrocytic VRAC channels attenuate glutamatedependent neuronal excitability and protect mice from brain damage. 15,16 In addition to their role in regulating cellular swelling, VRAC channels have also been implicated in cell apoptosis, insulin secretion, and antiviral immunity. 1,3,17 DCPIB is a commonly used specific inhibitor of VRAC channels, with an IC 50 of 4.1 μM in CPAE cells. 18 It has been proved that DCPIB has beneficial effects on reducing infarct size in middle cerebral artery occlusion (MCAO), 19 preventing swelling-induced shortening of guinea-pig atrial action potential duration, 18 and attenuating inflammatory response and neuronal injury in microglia. 20 Although other potent inhibitors of VRAC channels, such as pranlukast, zafirlukast, and 6u are available, 21,22 fewer inhibitors of VRAC channels are derived from natural product sources.
Syringin exhibits various pharmacological properties, including antioxidation, anti-inflammation, and immune inhibition. [23][24][25] It has been shown to reduce infarct volume and water content in MCAO animal models. 26 However, it is not clear whether syringin regulates VRAC channels.
In the present study, we recorded the endogenous VRAC currents induced by hypotonic extracellular solution in HEK293 cells.
We measured the VRAC currents before and after adding syringin at a potential of −80 mV. Additionally, we utilized molecular docking to predict the potential binding sites of the syringin.

| Reagents
Syringin (CAS: 118-34-3, HPLC >98%) was purchased from Herbpurify, dissolved in DMSO to make a stock solution, and stored at −20°C. Solutions were freshly prepared from stock solutions before each experiment and protected from light. The final concentration of DMSO was less than 0.1%.

| Electrophysiological recording
Volume regulated anion channel currents were recorded in the standard whole-cell configuration at 15-25°C using EPC 10 USB patch-clamp amplifier and PATCHMASTER NEXT software (HEKA, GER). The sampling rate was 20 kHz and digitally filtered at 2 kHz.
Series resistance compensation was set to 60%-80%. The electrodes were pulled by SUTTER P-1000 puller, and the resistance was maintained at 3-5 MΩ. In the patch-clamp experiments, bathing solution perfusion was delivered by eight-channel perfusion valve control system (Warner Instruments).
The standard test protocol consisted of a 600 ms step to −80 mV followed by a 2600 ms ramp from −100 to +100 mV from a holding potential of −30 mV, applied at 1500 ms intervals. The full voltage protocols consisted of a 2000 ms step protocol from −120 to +120 mV in 20 mV increment from a holding potential of −80 mV applied every 5000 ms.

| Molecular docking
The 3D structure of the VRAC protein (PDB number: 6NZW) was obtained from the RCSB PDB Protein Data Bank (https://www.rcsb. org/). The solvent and organic were removed from VRAC protein by PyMOL software. The 2D structure of syringin (PubChem CID: 5316860) was acquired from PubChem (https://pubch em.ncbi.nlm. nih.gov/). The energy of syringin was reduced by the MM2 method in Chem 3D 19.0. The charge was added to syringin by Autodock Vina MGLTools 1.5.6. software. 27 DCPIB is a VRAC channel specific inhibitor, the binding pocket of which was wrapped up by the grid box (center_x = 199, center_y = 199, center_z = 135, size_x = 10, size_y = 10, size_z = 18). The molecular docking work of syringin and VRAC protein depends on the grid box. The energy range was set as 1. The potential interacting amino acid residues were predicted by PyMOL software.

| Statistical analysis
Images were integrated by Adobe Illustrator CS5 software. The original data analysis software is Graphpad prism 7.0. All experimental data are presented as mean ± standard error (mean ± SEM).

| Nomenclature of target and ligand
Key protein target and ligand in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 28 and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20. 29

| The inhibition of syringin on VRAC channels
Syringin is a monosaccharide derivative, composed of trans-sinapyl alcohol that is linked to a β-D-glucopyranosyl residue at position 1 through a glycosidic linkage (Figure 1). It has been found to have potential for rescuing MCAO animal models. However, the regulatory function of syringin on VRAC channels is still unknown.
The patch-clamp technique is considered the gold standard for assessing ion channel functionality. In order to investigate the effect of syringin on VRAC channels, we used the whole-cell patch-clamp method to measure changes in VRAC currents as a detection index. As ( Figure 2D,F). Taken together, these results suggested that syringin suppressed VRAC channels in a dose-dependent relationship. Our study first identified syringin as a VRAC channels blocker. In total, nine combinations of syringin and VRAC were calculated by the software. Among them, the best conformation was predicted to have a binding affinity of −6.8 kcal/mol ( Table 1).

| DISCUSS ION
In conclusion, our study investigated the effects of syringin on en- of VRAC current. Additionally, in silico calculations indicated that syringin may have a high affinity for LRRC8/VRAC protein, suggesting that syringin may have a potential direct binding effect on VRAC channels. Given that VRAC channels mediate the osmotic excitotoxic substances efflux from astrocytes in ischemic injury, we propose that syringin may have the potential to rescue swollen astrocytes in ischemic injury by inhibiting VRAC channels, which could reduce the release of glutamate and potentially decrease neurotoxic damage to neurons. Syringin has been shown to possess anti-oxidant properties, as evidenced by its ability to inhibit reactive oxygen species (ROS) levels in the brains of APP/PS1 transgenic mice, 31 reduce mtROS induced by Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) Interestingly, 6u also acts as a dual inhibitor, as it is able to inhibit TREK1 channels. 21 In summary, VRAC channels lack selective regulators, which limits further research on the VRAC channels. For this reason, we need to discover regulators with another whole new structure.
In the current study, we first characterize syringin as an inhibitor for the VRAC channels. Syringin is the first VRAC regulator with a monosaccharide derivative structure, which provides a perspective for future VRAC channel regulator development. We hope that the inhibitory effect of syringin on VRAC channels will provide a theoretical basis for the inhibitory mechanism of syringin on brain edema.

AUTH O R CO NTR I B UTI O N S
Participated in research design and performance: Ziwei Xu, Wei Yao, Minyan Liang, Song Wang, Longhui Lu, Jingjing Wang, and Na Zhu.
Wrote or contributed to the writing of the manuscript: Ziwei Xu and Liping Huang.

ACK N OWLED G M ENT
The authors would like to acknowledge Dr. Liping Huang for her invaluable scientific discussion and generous help with experimental troubleshooting.

FU N D I N G I N FO R M ATI O N
We gratefully acknowledge the financial support for this study provided in part by the National Natural Science Foundation of China

DATA AVA I L A B I L I T Y S TAT E M E N T
Raw data can be made available upon request from the authors.

D I SCLOS U R E
All authors declare that they have no conflict of interest.

E TH I C S A PPROVA L S TATE M E NT
None.
TA B L E 1 Docking parameters of syringin and volume regulated anion channel protein.